Elucidating Pattern Formation in Anabaena: A Genetic Approach

在丝状蓝藻中的鱼腥藻，它通过沿着专门用于固定氮气的丝状半规则间隔的细胞(异形囊)精细地形成对氮素缺乏的反应。异形囊的模式是由发育和成熟的异形囊发出的抑制场启动和维持的。沃尔克博士分离了发育早期有缺陷的突变体，并发现了补充其中某些突变体的宇宙体。为了研究发育中的细丝基因表达的空间方面，他开发了一种载体，可以促进荧光素酶(Lux)的转录融合及其在鱼腥藻中的维持。他建议：1.进一步开发和表征Lux用于监测单个细胞的基因表达。2.确定一个在发育早期表达的基因，特别是在注定要成为异形囊的细胞中。候选启动子将被融合到勒克斯，并在整个开发过程中监测长丝的发光。3.对携带成功启动子/lux融合的鱼腥藻进行转座子诱变和反义RNA表型突变，寻找调控启动子表达的基因。这些基因将通过改变光发射来识别。沃尔克博士将专注于通过在所有细胞中诱导光发射来响应N-步降的突变体，而不仅仅是在发育中的异形囊中。4.克隆影响启动子表达的基因，并将其与Lux融合，在时间和空间上定位其表达。高等真核生物从单个细胞发展到各种类型和功能的细胞集合，但原核生物中的非均质分化很少见。沃尔克博士建议利用丝状蓝藻鱼腥藻的分化，在一个简单的原核生物中研究这些过程，在那里可以利用强大的基因技术。

Anabaena in a filamentous cyanobacterium that responds to nitrogen deprivation by elaborating along the filament semi-regularly spaced cells (heterocysts) specialized for N2-fixation. The pattern of heterocysts is initiated amd maintained by an inhibitory field emanating from developing and mature heterocysts. Dr. Wolk has isolated mutants that are defective early in development and have found cosmids that complement certain of these mutants. To study spatial aspects of gene expression in the developing filament, he has developed a vector that facilitates transcriptional fusions to luciferase (lux) and their maintenance in Anabaena. He proposes to: 1. Further develop and characterize the use of lux to monitor gene expression from individual cells. 2. Identify a gene that is expressed early in development, specifically in cells fated to become heterocysts. Candidate promoters will be fused to lux and light emission along the filament monitored through the course of development. 3. Subject Anabaena carrying the successful promoter/lux fusions to transposon mutagenesis and antisense RNA phenotypic mutagenesis to search for genes that regulate the expression of the promoter. Such genes will be identified by altered light emission. Dr. Wolk will focus on mutants that respond to N- stepdown by inducing light emission in all cells rather than only in developing heterocysts. 4. Clone genes that affect the expression of the promoter and, by fusing them to lux, localize their expression in time and space. Higher eukaryotes develop from single cells to collections of cells of varied types and functions, but inhomogeneous differentiation in prokaryotes is rare. Dr. Wolk proposes to exploit the differentiation of the filamentous cyanobacterium, Anabaena, to study these processes in a simple prokaryote where powerful genetic techniques can be utilized.